Site-specific nucleases are powerful reagents for specifically and efficiently targeting and modifying a DNA sequence within a complex genome. The double-stranded DNA breaks caused by site-specific nucleases are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). Although homologous recombination typically uses the sister chromatid of the damaged DNA as a donor matrix from which to perform perfect repair of the genetic lesion, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the double strand break. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used for the creation of specific gene knockouts. There are numerous applications of genome engineering by site-specific nucleases extending from basic research to bioindustrial applications and human therapeutics. Re-engineering a DNA-binding protein for this purpose has been mainly limited to the naturally occurring LADLIDADG homing endonuclease (LHE), artificial zinc finger proteins (ZFP), the Transcription Activator Like Effectors nucleases (TALE-nucleases), and the recently described CRISPR-Cas system.
Homing endonucleases, also known as meganucleases, are sequence-specific endonucleases with large (>14 bp) cleavage sites that can deliver DNA double-strand breaks at specific loci (Thierry and Dujon 1992). There are a handful of known homing endonuclease families which are demarcated on the basis of canonical motifs and the structural features which comprise them. However, they all share the property of recognizing and cleaving long DNA targets. Homing endonucleases were the first, and to date only, naturally occurring endonucleases with specificities at or approaching ‘genome level’, meaning having putative target sequences that occur very infrequently, or perhaps singularly, in their host genome. As a general property, HEs have a moderate degree of fidelity to their DNA target sequences, such that most base pair substitutions to their DNA target sequences reduce or eliminate the ability of the HE to bind or cleave it. HEs are therefore the most specific naturally occurring endonucleases yet discovered, and indeed this property is critical to the natural life cycle of the genetic elements in which they are encoded.
Homing endonuclease genes (HEGs) are classified as a type of selfish genetic element, as their DNA recognition and cleavage activity can lead to a DNA repair event that results in the copying of the HEG into the cleavage site. This mechanism of horizontal gene transfer, referred to as ‘homing’ results in a super-Mendelian inheritance pattern. Using this mechanism, HEGs and their endonuclease gene products can spread rapidly within their host species populations, and have also spread throughout all kingdoms of life over evolutionary time. HEGs are most commonly found in highly conserved genomic locations that do not impart fitness costs on their host organisms, such as within introns or as non-disruptive N- or C-terminal fusions to host proteins.
The LAGLIDADG homing endonuclease family (LHE) comprises a group of compact (<320 amino acids) nucleases whose structural and mechanistic properties have been studied extensively owing to their attractive properties for genome engineering applications. LHEs operate either as dimers or as pseudo-dimeric monomers, with the DNA cleaving active site occurring at the DNA-facing end of the interface of the two subunits (in dimeric LHEs) or domains (in monomeric LHEs). The LAGLIDADG consensus motifs for which LHEs are named are found in the two central alpha helices which form this interface between the two subunits or domains. At the bottom of each LAGLIDADG helix are the residues which together coordinate the hydrolysis reaction if the appropriate conditions are met, such as if the LHE finds and binds to an appropriate DNA target sequence. The active site covers the ‘central-4’ DNA bases of the DNA target sequence.
On either side of the active site are the two DNA binding domains LHEs use to recognize their DNA target sequences. Each domain comprises an anti-parallel beta sheet which wraps around nearly a complete turn of DNA and contacts 9 base pairs of DNA sequence. Members of the LHE family thus recognize 22 base pair DNA target sequences (9 base pairs for each domain, and 4 base pairs covered by the active site), which are partially palindromic in the case of dimeric LHEs, but can be entirely asymmetric for monomeric LHEs. Emanating from each anti-parallel beta sheet are the amino acid side chains which comprise the DNA recognition interface. While there is much amino acid conservation throughout the non-DNA interfacing residues amongst the LHE family, DNA recognition interface amino acid compositions vary significantly. This is because for each LHE the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which is necessarily unique to a particular LHE's DNA target sequence. The amino acid composition of the DNA recognition interface (and the correspondence of it to a particular DNA sequence) is therefore the definitive feature of any natural or engineered LHE. The DNA recognition interface functions in determining the identity of the DNA target sequence which can be accommodated and hydrolyzed and also the affinity and specificity properties which define the quality of the LHE according to the demands of the application.
Owing to their small size and exquisite specificity properties, LHEs have been the subject of numerous efforts to engineer their DNA recognition properties with the desired outcome of cleaving and altering genes of interest in research, biotechnology, crop science, global health, and human therapeutics applications. However, the extent of the networks of residues which form the DNA recognition interface has generally prevented efficient methods for re-addressing LHEs to DNA target sequences of interest. This has led to continued innovation in field of gene-specific nuclease engineering, with three endonuclease alternative platforms now validated as having the capacity to target DNA sequences with ranging (but generally high) levels of specificity, as well as new and improved methods for overcoming the challenges of engineering the DNA recognition interfaces of LHEs.
Zinc finger nucleases (ZFNs) generating by fusing a plurality of Zinc finger-based DNA binding domains to an independent catalytic domain (Kim, Cha et al. 1996; Smith, Berg et al. 1999; Smith, Bibikova et al. 2000) represent another type of engineered nuclease commonly used to stimulate gene targeting and have been successfully used to induce gene correction, gene insertion and gene deletion in research and therapeutic applications. The archetypal ZFNs are based on the catalytic domain of the Type IIS restriction enzyme FokI and Zinc Finger-based DNA binding domains made of strings of 3 or 4 individual Zinc Fingers, each recognizing a DNA triplet (Pabo, Peisach et al. 2001). Two Zinc Finger-FokI monomers have to bind to their respective Zinc Finger DNA-recognition sites on opposite strands in an inverted orientation in order to form a catalytically active dimer that catalyze double strand cleavage (Bitinaite, Wah et al. 1998).
Transcription activator-like effectors (TALEs) were the next artificial endonuclease platform. TALEs derived from a family of proteins used in the infection process by plant pathogens of the Xanthomonas or Ralstonia genus are repetitive proteins characterized by 14-20 repeats of 33-35 amino acids differing essentially by two positions. Each base pair in the DNA target is contacted by a single repeat, with the specificity resulting from the two variant amino acids of the repeat (the so-called repeat variable dipeptide, RVD). The apparent modularity of these DNA binding domains has been confirmed to a certain extent by modular assembly of designed TALE-derived protein with new specificities (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). Very similarly to ZFNs, TALEs were readily adapted into site-specific nucleases by arraying TALE repeats with RVDs corresponding to the target sequence of choice and fusing the resultant array to a FokI domain. As such, DNA cleavage by a TALE-Nuclease requires two DNA recognition regions flanking an unspecific central region. TALE nucleases have proliferated widely since 2010 owing to their ease of production and improved double-strand break generating efficiency.
Of these distinct technologies, it is important to distinguish the advantaged properties of each and to determine innovative ways to capture these properties for the appropriate genome engineering applications. One of the most powerful applications of site-specific nuclease technology is in the field of human therapeutics, which requires the use of highly efficient and specific nuclease reagents to safely and effectively edit genomic information in human cells or tissues. A one prominent example is the cancer immunotherapy field, which is at the forefront of applying nuclease technological advances for developing novel therapeutics. These approaches are focused on harnessing the powerful anti-tumor activities of patient-derived (autologous) or donor-derived (allogeneic) T-cells and leveraging this potential via genome engineering of cell-intrinsic properties such as cellular proliferation, engraftment, migration or longevity. Successful and scalable manufacture of T-cells endowed with enhanced anti-cancer activity requires generation of highly efficient nuclease compositions and simplified delivery strategies, such as those described in some aspects of this application.
The immune system has a key foundational role in detecting and preventing the development of human cancer. The majority of transformed cells are quickly detected by immune sentinels and destroyed through the activation of antigen-specific T-cells via clonally expressed T-cell receptors (TCR). Oncogenesis is thus an immunological disorder, a failure of immune system to mount the necessary anti-tumor response to durably suppress and eliminate the disease. Certain immunotherapy interventions developed over the last few decades, such as recombinant cytokine infusions, have specifically focused enhancing T-cell immunity, and while these have been associated with sporadic cases of disease remission, they have not had substantial overall success. Recent therapies with monoclonal antibodies targeting molecules which inhibit T-cell activation, such as CTLA-4 or PD-1, have shown a more substantial anti-tumor effect, however these treatments are associated with substantial toxicity due to systemic immune activation. Most recently, therapeutic strategies which are based on the isolation, modification, expansion and reinfusion of T-cells have been explored and tested in early stage clinical trials. These treatments have shown mixed rates of success, but a number of patients have experienced unprecedented objective responses and durable remissions, highlighting the potential for T-cell based cancer immunotherapies. Genome editing strategies which are designed to harness this potential for successful widespread implementation of T-cell cancer immunotherapies are described herein.
Successful recognition of tumor cell associated antigens by cytolytic T-cells initiates targeted tumor lysis and underpins any effective cancer immunotherapy approach. Some tumors contain tumor-infiltrating T-cells (TILs) which express TCRs specifically directed tumor-associated antigens; however access to substantial numbers of TILs is limited to only a few human cancers. In response to this limitation, artificial antigen recognition and signaling transgenes called chimeric antigen receptors (CARs) have been devised to broaden the scope and utility of T-cell based cancer immunotherapy. CARs are transmembrane spanning proteins whose extracellular portions contain antigen recognition domains most typically derived from single-chain variable fragments (scFv) of monoclonal antibodies, and whose intracellular domains contain combinations of signaling domains to mimic TCR-like activation signals. It has been widely demonstrated that primary human T-cells made to express CARs are able to respond to and kill cells which bear the antigen recognized by the scFv domain.
Despite highly promising initial results with CAR-expressing transgenic T-cells, the efficacy, safety and scalability of CAR-based T-cell immunotherapies is limited by continuous expression of clonally derived TCR. Residual TCR expression may interfere with CAR signaling in engineered T-cells or it may initiate off-target and pathologic responses to self- or allo-antigens. Consequently, CAR-based T-cells have only been used in autologous applications. Genetic abolition of endogenous TCR through nuclease-mediated gene editing would reduce the risk of damaging collateral responses and decrease the potential for T-cell mediated graft vs. host disease (GVHD). The main hurdle for developing universal allogeneic T-cell therapy is the development of GVHD through the activation of donor T cells' TCR by the recipients' HLA complex. Removal of the TCR would prevent such graft-versus-host responses and enable the development of simple and widely applicable allogeneic T-cell therapies.
In addition to cancer, T-cell therapies are being developed for a wide range of therapeutic applications including chronic viral infections, autoimmune disease and stem cell transplantation. In disease models and initial clinical models have shown a key role for the regulatory T cell subset (T-regs) in controlling the development and extent of GVHD and various autoimmune diseases. Transfer of regulatory T cells ameliorates GVHD in patients receiving stem cell transplant. In addition, transfer of regulatory T cells improved disease outcome in preclinical models of rheumatoid arthritis, type-1 diabetes and systemic lupus erythematosus, amongst others. This approach is also being tested in patients with chronic viral infections such as Hepatitis B (HBV). Engineered T cells containing HBV-specific CARs are highly active against HBV-infected cells. These approaches are being tested in clinical trials, however their use is limited by the same manufacturing and scalability hurdles associated with other autologous therapies. Combining genetic targeting of TCR-alpha in T cells with CARs targeting tolerance or viral targets represents a very powerful way to develop allogeneic T cell therapy for the treatment of human disease.